Angiogensis is essential for wound healing. Deficient or exuberant angiogenesis, however, contributes to pathogenesis of cutaneous ulcers, ischemic injury, psoriasis, arthritis, vascular sclerosis, as well as the ocular and non-ocular complications of diabetes. Several proteolytic systems regulate the angiogenic process. Gene knockout and reconstitution studies have identified plasminogen activator inhibitor type-I (PAI-I), the major physiologic regulator of plasmin activation, as essential for neovascularization. PAI-I protects the stroma from excessive proteolysis, facilitates endothelial cell locomotion via regulation of cell-to-matrix adhesion and stabilizes nascent vessel structure. PAI-I transcription during endothelial cell activation and in vitro angiogenesis requires mitogen/ extracelIular-regulated kinase (MEK) activity and involves binding of the helix-loop-helix transcription factor USF-1 to an E box motif (CACGTG). We propose to test the hypothesis that mitogen-activated protein (MAP) kinase-mediated USF-1 phosphorylation stimulates E box-dependent DNA binding and regulates PAI-I transcription as part of the switch to angiogenic phenotype. The following Aims will be addressed: 1. We will determine the requirements (i.e., USF-1 transcript expression, protein synthesis, nuclear translocation, phosphorylation) for USF-1 occupancy of the PAI-I box site and induced PAI-I transcription during endothelial growth activation and, migration. 2. We will clarify the MAP kinase type-specificity for USF-1 binding/ phosphorylation and identify the specific residues in USF-I that are phosphorylated by MAP kinases. 3. We will ascertain if the PAI-I E box is an expression "modulating" motif acting as a platform for replacement of the PAI-I transcriptional inhibitor USF-2a with a USF-1 containing complex during the switch from a quiescent to activated endothelial phenotype. 4. We will utilize genetic constructs that target PAI-I transcripts and modulate USF-1 function to assess effects of PAI-I expression perturbation on endothelial cell migration, proliferation and capillary structure. This work will provide information necessary to fashion targeted therapeutic strategies to treat pathologic angiogenesis.